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• W2079782835 abstract "Müllerian inhibiting substance (MIS) inhibits breast cancer cell growth in vitro through interference with cell cycle progression and induction of apoptosis, a process associated with NFκB activation and up-regulation of one of its important target genes, IEX-1S (Segev, D. L., Ha, T., Tran, T. T., Kenneally, M., Harkin, P., Jung, M., MacLaughlin, D. T., Donahoe, P. K., and Maheswaran, S. (2000) J. Biol. Chem. 275, 28371–28379). Here we demonstrate that MIS activates the NFκB signaling cascade, induces IEX-1S mRNA, and inhibits the growth of MCF10A, an immortalized human breast epithelial cell line with characteristics of normal cells. In vivo, an inverse correlation was found to exist between various stages of mammary growth and MIS type II receptor expression. Receptor mRNA significantly diminished during puberty, when the ductal system branches and invades the adipose stroma and during the expansive growth at lactation, but it was up-regulated during involution, a time of regression and apoptosis. Peripartum variations in MIS type II receptor expression correlated with NFκB activation and IEX-1S mRNA expression. Administration of MIS to female mice induced NFκB DNA binding and IEX-1S mRNA expression in the breast. Furthermore, exposure to MIS in vivo increased apoptosis in the mouse mammary ductal epithelium. Thus, MIS may function as an endogenous hormonal regulator of NFκB signaling and growth in the breast. Müllerian inhibiting substance (MIS) inhibits breast cancer cell growth in vitro through interference with cell cycle progression and induction of apoptosis, a process associated with NFκB activation and up-regulation of one of its important target genes, IEX-1S (Segev, D. L., Ha, T., Tran, T. T., Kenneally, M., Harkin, P., Jung, M., MacLaughlin, D. T., Donahoe, P. K., and Maheswaran, S. (2000) J. Biol. Chem. 275, 28371–28379). Here we demonstrate that MIS activates the NFκB signaling cascade, induces IEX-1S mRNA, and inhibits the growth of MCF10A, an immortalized human breast epithelial cell line with characteristics of normal cells. In vivo, an inverse correlation was found to exist between various stages of mammary growth and MIS type II receptor expression. Receptor mRNA significantly diminished during puberty, when the ductal system branches and invades the adipose stroma and during the expansive growth at lactation, but it was up-regulated during involution, a time of regression and apoptosis. Peripartum variations in MIS type II receptor expression correlated with NFκB activation and IEX-1S mRNA expression. Administration of MIS to female mice induced NFκB DNA binding and IEX-1S mRNA expression in the breast. Furthermore, exposure to MIS in vivo increased apoptosis in the mouse mammary ductal epithelium. Thus, MIS may function as an endogenous hormonal regulator of NFκB signaling and growth in the breast. Müllerian inhibiting substance recombinant human MIS polymerase chain reaction nuclear factor κB The importance of MIS,1a sexually dimorphic member of the transforming growth factor β family of hormones, in regression of the Müllerian duct in male embryos is well established. MIS is produced by Sertoli cells of the testis even after regression of the Müllerian duct and continues to be made throughout adulthood. In females, synthesis by granulosa cells of the ovary commences after birth and persists until menopause (2Hudson P.L. Dougas I. Donahoe P.K. Cate R.L. Epstein J. Pepinsky R.B. MacLaughlin D.T. J. Clin. Endocrinol. Metab. 1990; 70: 16-22Crossref PubMed Scopus (224) Google Scholar, 3Lee M.M. Donahoe P.K. Hasegawa T. Silverman B. Crist G.B. Best S. Hasegawa Y. Noto R.A. Schoenfeld D. MacLaughlin D.T. J. Clin. Endocrinol. Metab. 1996; 81: 571-576Crossref PubMed Scopus (388) Google Scholar). The continued production of MIS throughout adolescence and adulthood in males and females implies other functional roles for this hormone after causing regression of the Müllerian duct. The MIS type II receptor, a highly conserved single transmembrane serine threonine kinase, is homologous to members of the transforming growth factor β family of type II receptors (4Baarends W.M. van Helmond M.J. Post M. van der Schoot P.J. Hoogerbrugge J.W. de Winter J.P. Uilenbroek J.T. Karels B. Wilming L.G. Meijers J.H. Themmen P.N. Grootegoed J.A. Development. 1994; 120: 189-197Crossref PubMed Google Scholar, 5di Clemente N. Wilson C. Faure E. Boussin L. Carmillo P. Tizard R. Picard J.Y. Vigier B. Josso N. Cate R. Mol. Endocrinol. 1994; 8: 1006-1020Crossref PubMed Scopus (199) Google Scholar, 6Teixeira J. He W.W. Shah P.C. Morikawa N. Lee M.M. Catlin E.A. Hudson P.L. Wing J. Maclaughlin D.T. Donahoe P.K. Endocrinology. 1996; 137: 160-165Crossref PubMed Scopus (95) Google Scholar). The binding of MIS ligand to its receptor initiates a signaling cascade that is dependent on recruitment of type I receptors, ALK2 and ALK6, which also signal for bone morphogenetic proteins (7Visser J.A. Olaso R. Verhoef-Post M. Kramer P. Themmen A.P.N. Ingraham H.A. Mol. Endocrinol. 2001; 15: 936-945Crossref PubMed Scopus (143) Google Scholar, 8Gouedard L. Chen Y.G. Thevenet L. Racine C. Borie S. Lamarre I. Josso N. Massague J. di Clemente N. J. Biol. Chem. 2000; 275: 27973-27978Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar, 9Clarke T.R. Hoshiya Y. Yi S.E. Liu X. Lyon K.M. Donahoe P.K. Mol. Endocrinol. 2001; 15: 946-959Crossref PubMed Scopus (157) Google Scholar). The MIS type II receptor gene contains 11 exons and encodes a 1.8-kilobase mRNA. It is expressed at high levels in the Müllerian duct, the Sertoli and granulosa cells of embryonic and adult gonads (6Teixeira J. He W.W. Shah P.C. Morikawa N. Lee M.M. Catlin E.A. Hudson P.L. Wing J. Maclaughlin D.T. Donahoe P.K. Endocrinology. 1996; 137: 160-165Crossref PubMed Scopus (95) Google Scholar). However, the status of receptor expression and MIS responsiveness in other tissues has yet to be clarified. Using several different techniques, we recently demonstrated MIS type II receptor expression in normal breast, human breast fibroadenomas, ductal carcinomas, and cancer cell lines (10Segev D.L. Ha T.U. Tran T.T. Kenneally M. Harkin P. Jung M. MacLaughlin D.T. Donahoe P.K. Maheswaran S. J. Biol. Chem. 2000; 275: 28371-28379Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar). MIS inhibited the growth of both estrogen receptor-positive and estrogen receptor-negative human breast cancer cells in vitro by interfering with cell cycle progression and inducing apoptosis. The effect of MIS on breast cell proliferation correlated with its ability to induce the NFκB family of transcription factors and to up-regulate IEX-1S (10Segev D.L. Ha T.U. Tran T.T. Kenneally M. Harkin P. Jung M. MacLaughlin D.T. Donahoe P.K. Maheswaran S. J. Biol. Chem. 2000; 275: 28371-28379Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar), an immediate early gene known to be induced following NFκB activation by other extracellular signals. PRG1 and gly96 represent the rat and mouse homologues, respectively, of human IEX-1 (11Schafer H. Diebel J. Arlt A. Trauzold A. Schmidt W.E. FEBS Lett. 1998; 436: 139-143Crossref PubMed Scopus (62) Google Scholar). Overexpression of IEX-1S in breast cancer cells inhibited their growth, indicating a negative growth regulatory role for this newly identified NFκB-inducible gene (10Segev D.L. Ha T.U. Tran T.T. Kenneally M. Harkin P. Jung M. MacLaughlin D.T. Donahoe P.K. Maheswaran S. J. Biol. Chem. 2000; 275: 28371-28379Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar). The NFκB family consists of transcriptional activators, including p65, p50, p52, and c-Rel, that share a Rel homology domain and form either homo- or heterodimers that bind to DNA in a sequence-specific manner. NFκB in its inactive state exists in the cytosol bound to the inhibitory IκB family of molecules. Activation of the pathway by extracellular signals leads to phosphorylation and degradation of IκB with subsequent nuclear localization of NFκB (12Baichwal V.R. Baeuerle P.A. Curr. Biol. 1997; 7: R94-R96Abstract Full Text Full Text PDF PubMed Google Scholar, 13Barkett M. Gilmore T.D. Oncogene. 1999; 18: 6910-6924Crossref PubMed Scopus (1082) Google Scholar). Expression of a dominant negative inhibitor of NFκB (IκBα-DN) in breast cancer cells ablated MIS-mediated induction of IEX-1S, inhibition of growth, and induction of apoptosis, indicating that activation of the NFκB pathway was required for these processes (10Segev D.L. Ha T.U. Tran T.T. Kenneally M. Harkin P. Jung M. MacLaughlin D.T. Donahoe P.K. Maheswaran S. J. Biol. Chem. 2000; 275: 28371-28379Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar). In order to determine whether MIS-mediated growth inhibition through activation of the NFκB signaling cascade, previously characterized using human breast cancer cell lines, is also functional in normal breast tissue, we analyzed the effect of MIS on MCF10A, a nontumorigenic breast epithelial cell line (1Soule H.D. Maloney T.M. Wolman S.R. Peterson W.D. Brenz R. McGrath C.M. Russo J. Pauley R.J. Jones R.F. Brooks S.C. Cancer Res. 1990; 50: 6075-6086PubMed Google Scholar), and on murine mammary glands in vivo. Furthermore, to evaluate whether these events are developmentally regulated, we analyzed endogenous MIS type II receptor expression, NFκB activity, and IEX-1 expression in the mammary gland during postnatal morphogenesis. In this report, we demonstrate that MIS activates the NFκB signaling cascade, induces IEX-1S expression, and inhibits the growth of MCF10A cells. Peripartum expression of MIS type II receptor in the rat breast correlated with the level of NFκB DNA binding activity and expression of IEX-1S mRNA. In addition, exogenous MIS activated NFκB DNA binding and induced IEX-1S expression in the mammary glands of adult mice and increased the number of apoptotic cells in the ductal epithelium of the breast in vivo. Thus, MIS might be a hormonal regulator of the NFκB signaling cascade in vivo and a negative regulator of normal breast growth. Human breast cancer cell line T47D was grown in Dulbecco's modified medium supplemented with 10% female fetal bovine serum, glutamine, and penicillin/streptomycin. MCF10A cells were grown in mammary epithelial growth medium (Clonetics) supplemented with 100 ng/ml cholera toxin (Calbiochem). 184A1 cells (a gift from Dr. Martha Stampfer) were grown in mammary epithelial growth medium supplemented with isoproterenol and transferrin. To test the growth inhibitory effect of exogenous MIS, MCF 10A cells were seeded in 100-mm tissue culture flasks in the absence of MIS. MIS was added after 24 h, cultures were grown in the presence or absence of 35 nm MIS for 3 days, and cell numbers were compared by Coulter counter. MIS type II receptor expression analyses in the rat breast during development and peripartum stages were done using Harlan Sprague-Dawley rats. Recombinant human MIS (rhMIS) was collected from growth medium of Chinese hamster ovary cells transfected with the human MIS gene and purified as described in Ref. 14Ragin R.C. Donahoe P.K. Kenneally M.K. Ahmad M.F. MacLaughlin D.T. Protein Expression Purif. 1992; 3: 236-245Crossref PubMed Scopus (61) Google Scholar. To study the effects of rhMIS on the mammary gland, adult female C3H mice (8 weeks old; average weight, 25 g) were obtained from the Edwin L. Steele Laboratory, Massachusetts General Hospital (Boston, MA). All animals were cared for and experiments performed in this facility under guidelines approved by the Assessment and Accreditation of Laboratory Animal Care using protocols approved by the Institutional Review Board-Institutional Animal Care and Use Committee of the Massachusetts General Hospital. All experiments were performed using ketamine/xylazine (100/10 mg/kg) for anesthesia. Each animal was injected intraperitoneally with 100 µg of rhMIS or phosphate-buffered saline (vehicle control). Breast tissue was harvested bilaterally from each animal for RNA isolation and gel shift assays. Blood was drawn from the animals at the time of tissue harvest to determine the circulating level of rhMIS using MIS-enzyme-linked immunosorbent assay. Six-week-old female Rag 2 knockout mice (16Shinkai Y. Rathbun G. Lam K.P. Oltz E.M. Stewart V. Mendelsohn M. Charron J. Datta M. Young F. Stall A.M. Alt F.W. Cell. 1992; 68: 855-867Abstract Full Text PDF PubMed Scopus (2199) Google Scholar) were injected intraperitoneally with 100 µg of rhMIS (n = 4) or vehicle (PBS, n = 4) twice daily for 7 days. At the end of this period, breast tissue was harvested bilaterally from each animal, and serum was collected to determine the circulating levels of rhMIS. Part of the tissue was embedded in paraffin for ApopTag assay and fluorescein-labeled in situ cell death detection. MCF10A and T47D cells were grown to 70% confluence and treated with 35 nm rhMIS for 1 h. Cells were harvested in cold PBS, resuspended in 1 ml of TKM (10:10:1) (10 mm Tris, pH 8.0, 10 mm KCl, and 1 mm MgCl2), and lysed with 0.1% Triton X-100. Nuclei were pelleted by centrifugation at 5000 rpm at 4 °C, and proteins were extracted in buffer containing 10 mm HEPES, pH 7.0, 350 mm NaCl, and 1 mm EDTA. 3 µg of protein was used in 25-µl binding reactions containing 10 mm HEPES, pH 7.0, 70 mm NaCl, 0.1% Triton X-100, and 4% glycerol. The oligonucleotide containing the consensus DNA binding sequence for NFκB proteins (Promega) was 32P-end-labeled, and DNA-protein complexes were resolved on 4% native polyacrylamide gels. Supershift experiments were performed by adding 0.1 µg of rabbit anti-p65 or p50 antibodies (Santa Cruz Biotechnology) to the binding reactions. Nuclear protein extraction from tissues was performed as described by Sovak et al. (27Sovak M.A. Bellas R.E. Kim D.W. Zanieski G.J. Rogers A.E. Traish A.M. Sonenshein G.E. J. Clin. Invest. 1997; 100: 2952-2960Crossref PubMed Scopus (651) Google Scholar). RNase protection assays to detect MIS type II receptor expression were done as previously described (10Segev D.L. Ha T.U. Tran T.T. Kenneally M. Harkin P. Jung M. MacLaughlin D.T. Donahoe P.K. Maheswaran S. J. Biol. Chem. 2000; 275: 28371-28379Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar). To generate a riboprobe to detect MIS type II receptor expression in the rat, a DNA fragment containing part of exon 11 and the 3′ untranslated region of the rat MIS type II receptor was amplified using the following primers: sense, 5′-CCCCGAATTCCCTGGCTTATCCTCAGG-3′; antisense, 5′-CCCCCTCGAGTCAGCCTGTACAGAGTTCATATGA-3′. The rat MIS type II receptor cDNA was used as the template. The receptor fragment was cloned in reverse orientation into XhoI-EcoRI sites of the pCDNA 3.1(–) plasmid. The resulting construct was sequenced to confirm the boundaries of the insert and linearized withHindIII, and the antisense transcript was obtained using T7 polymerase (MAXIscript in vitro transcription kit, Ambion). RNase protection assays were done with 90–100 µg of total RNA isolated from the rat breast at various stages of development using the RPA III ribonuclease protection assay kit (Ambion). RNA samples derived from both individual animals or pooled from groups of three animals were analyzed as indicated in the figure legends. Briefly, RNA was hybridized with 75–80 pg of radiolabeled probe overnight at 50–55 °C and digested with a mixture of RNase A and RNase T1 for 30 min at 37 °C. The protected fragments were precipitated and analyzed on a denaturing 6% polycrylamide/6 m urea gel. The same amount of yeast tRNA were used as a positive control for the function of RNase, and another sample containing the same amount of yeast tRNA was incubated without RNase to control for probe integrity. The riboprobe to detect MIS type II receptor expression in the mouse was generated by PCR amplification using the following primers: sense, 5′-CCC CGA ATT CTG CCC AGA GAA CTC CCT T-3′; antisense, 5′-CCC CCT CGA GTT CCT GAG CAT ATC TAC CCC-3′. cDNA generated from RNA isolated from the mouse testis was used as the template. RNase protection was done as described above. The riboprobe to detect the long and short forms of PRG1/IEX-1 mRNA in the rat was generated by PCR amplification using the following primers: sense, 5′-AAC CAC CTC CAC ACC ATG ACT G-3′; antisense, 5′-CCT TCT TCA GCC ATC AAA ATC TGG-3′. Rat genomic DNA was used as the template. The resulting fragment was cloned into SrfI sites of the pPCR-script Amp sk (+) plasmid. The construct was linearized with SalI, and the antisense transcript was obtained with T3 polymerase (MAXIscript in vitro transcription kit, Ambion). RNase protection was done as described above. Primers for detecting MIS type II receptor expression in MCF 10A and 184A1 cells were as follows: sense, 5′-GCT GGC TTA TGC TCT TCT CCT TC-3′; antisense, 5′-ACC TCG CAC TCT GTA GTT CTT TCG-3′. Total RNA was converted to cDNA and amplified with the above primers by PCR. RNA was isolated from cells or tissue samples using RNA STAT-60 total RNA isolation kit (Tel-Test, Inc.). Indicated amounts of RNA were separated on a formaldehyde gel, transferred to HyBond membrane (Amersham Pharmacia Biotech), and probed with human IEX-1 or mouse gly96/IEX-1 as indicated in the figure legends. The human IEX-1 probe for Northern analysis was derived by PCR amplification as previously described (10Segev D.L. Ha T.U. Tran T.T. Kenneally M. Harkin P. Jung M. MacLaughlin D.T. Donahoe P.K. Maheswaran S. J. Biol. Chem. 2000; 275: 28371-28379Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar). The probe for detecting both the long and short forms of mouse gly96/IEX-1 was derived by PCR amplification using the following primers: sense, 5′-AAC CAC CTC CAC ACC ATG ACT G-3′; antisense, 5′-CCT TCT TCA GCC ATC AAA ATC TGG-3′. Primers for detecting the presence of IEX-1L and IEX-1S transcripts in MCF10A cells have been described (10Segev D.L. Ha T.U. Tran T.T. Kenneally M. Harkin P. Jung M. MacLaughlin D.T. Donahoe P.K. Maheswaran S. J. Biol. Chem. 2000; 275: 28371-28379Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar). Breast tissue was harvested, fixed, and embedded in paraffin. After sectioning and deparaffinization, apoptotic cells were detected using a fluorescein in situ cell death detection kit (Roche Molecular Biochemicals) as indicated in the user manual. Images were obtained at a magnification of × 60. For confirmation, the same tissue was sectioned and stained using an ApopTag peroxidase in situ apoptosis detection kit (Intergen) using the protocol provided in the user's manual. Number of apoptotic cells on each slide was compared with number of mammary ducts seen in cross section and expressed as a ratio normalized to the average ratio in the controls. We had previously demonstrated that MIS inhibits the growth of both estrogen receptor-positive and estrogen receptor-negative human breast cancer cells in vitro through activation of the NFκB signaling cascade. In order to determine whether MIS could also inhibit the growth of nontumorigenic breast epithelial cells, we analyzed MCF10A cells, a human mammary epithelial cell line with normal karyotype derived from a patient with fibrocystic breast disease (1Soule H.D. Maloney T.M. Wolman S.R. Peterson W.D. Brenz R. McGrath C.M. Russo J. Pauley R.J. Jones R.F. Brooks S.C. Cancer Res. 1990; 50: 6075-6086PubMed Google Scholar), as well as 184A1 cells (17Stampfer M.R. Bartley J.C. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 2394-2398Crossref PubMed Scopus (360) Google Scholar). Reverse transcription-PCR analysis demonstrated the presence of receptor in both MCF10A and 184A1 cells (Fig. 1 A). Sequence analysis of the 582-base pair DNA fragment was identical to exons 1, 2, 3, 4, and 5 of the human MIS type II receptor (data not shown). Treatment of MCA10A cells with MIS induced three NFκB DNA-protein complexes following 1 h of treatment (Fig. 1 B, left panel). The heavier complex comigrated with the NFκB DNA-protein complex stimulated in T47D cells following MIS treatment (Fig. 1 B, right panel). Simultaneous addition of either rabbit anti-p50 or anti-p65 antibodies to the binding reaction demonstrated that the heaviest complex consisted predominantly of p50 and p65 subunits, as was demonstrated in T47D cells (10Segev D.L. Ha T.U. Tran T.T. Kenneally M. Harkin P. Jung M. MacLaughlin D.T. Donahoe P.K. Maheswaran S. J. Biol. Chem. 2000; 275: 28371-28379Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar). The faster migrating complexes, however, were unique to MCF10A cells and contained p50 subunits. Incubation of nuclear lysates with anti-c-Rel antibody did not supershift the complexes, suggesting that c-Rel protein was not present in them We next investigated whether MIS-mediated increase in NFκB DNA binding activity in MCF10A cells correlated with the induction of its target gene IEX-1S. An estimated 5-fold induction of IEX-1 mRNA was observed following 1 h of treatment with MIS, suggesting that the NFκB binding activity induced by MIS was functionally active (Fig.1 C, top panel). Reverse transcription-PCR analysis of the MIS-treated samples demonstrated that MIS selectively up-regulated the IEX-1S transcript (Fig. 1 C, bottom panel). We had previously demonstrated that MIS inhibited the growth of breast cancer cellsin vitro through a NFκB-mediated mechanism. As with breast cancer cell lines, treatment of MCF10A cells in vitro with exogenous MIS inhibited growth by 60% (Fig. 1 D). To confirm that MIS-mediated signaling events and its effects on breast epithelial cell growth identified using thein vitro cell systems are functional in vivo, we investigated whether MIS type II receptor is expressed in the normal breast. Total RNA isolated from the mammary glands of 8-week-old mice was analyzed by RNase protection assay using an antisense riboprobe specific for exon 11 and 3′ untranslated region of the mouse MIS type II receptor (Fig. 2 A, left panel). The protected fragment was 89 base pairs shorter than the probe due to unrelated sequences at the 5′ and 3′ ends. Detection of a protected fragment of the expected size in the breast, which comigrated with that from the testis, confirmed that the MIS type II receptor mRNA was expressed in normal breast but at a level much lower than that in the testis (Fig. 2 A, right panel). MIS type II receptor was also detected in rat breast by RNase protection assay using an antisense riboprobe that contained exon 11 and the 3′ untranslated region specific to the rat MIS type II receptor DNA sequence. Because mammary tissue undergoes the majority of its development in the adult and robust expansion of the breast epithelium occurs during pregnancy and continues into lactation, MIS type II receptor mRNA levels were analyzed using total RNA isolated from mammary glands of virgin, pregnant, lactating, and weaned rats (Fig.2 B). Phosphorimaging of band intensities from three independent experiments demonstrated an 80% decrease in MIS type II receptor expression 2 days after delivery during early lactation. The receptor mRNA rebounded to higher levels 2 days after removal of pups, a period of ductal regression (Fig. 2 B, bottom panel). Analysis of MIS type II receptor expression during breast development in Harlan Sprague-Dawley rats revealed a gradual increase up to postnatal day 30 and a decrease in three individual animals older than 30 days (Fig. 2 C, top panel). Quantification of transcript levels by phophorimaging analysis demonstrated a 2.5-fold decrease in MIS type II receptor between animals of postnatal days 14–30 and postnatal days 40–60 (Fig. 2 C, bottom panel). Onset of pubertal changes in Harlan Sprague-Dawley rats occur at an average age of 35 days after birth (18McGivern R.F. Yellon S.M. Alcohol. 1992; 9: 335-340Crossref PubMed Scopus (35) Google Scholar, 19Juarez J. Barrios De Tomasi E. Vazquez C. Alcohol. 2000; 21: 181-185Crossref PubMed Scopus (7) Google Scholar). Interestingly, lowering of MIS type II receptor expression during breast development in the rat coincides with puberty, when the ductal system branches and invades the fat pad (20Masso-Welch P.A. Darcy K.M. Stangle-Castor N.C. Ip M.M. J. Mamm. Gland Biol. Neoplasia. 2000; 5: 165-185Crossref PubMed Scopus (117) Google Scholar). The inverse correlation between MIS type II receptor expression and growth in the breast during puberty and peripartum stages was compatible with the hypothesis that MIS-mediated signaling may exert an inhibitory effect on proliferation. Several transcription factors, including NFκB, are regulated during tissue remodeling of the breast. In mice, NFκB DNA binding activity in the breast increases slightly during pregnancy, with undetectable levels during lactation (21Clarkson R.W. Heeley J.L. Chapman R. Aillet F. Hay R.T. Wyllie A. Watson C.J. J. Biol. Chem. 2000; 275: 12737-12742Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar, 22Geymayer S. Doppler W. FASEB J. 2000; 14: 1159-1170Crossref PubMed Scopus (67) Google Scholar), and is robustly induced in the involuting mammary gland, with the highest levels of binding evident at 2–3 days of involution (21Clarkson R.W. Heeley J.L. Chapman R. Aillet F. Hay R.T. Wyllie A. Watson C.J. J. Biol. Chem. 2000; 275: 12737-12742Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar). In order to determine whether activation of NFκB DNA binding correlated with changes in MIS type II receptor expression during breast development in rats, electromobility gel shift assays were performed. NFκB DNA binding activity was quite readily detectable in the mammary glands of both virgin and pregnant rats. Consistent with the results in mice, reported by Clarkson et al. (21Clarkson R.W. Heeley J.L. Chapman R. Aillet F. Hay R.T. Wyllie A. Watson C.J. J. Biol. Chem. 2000; 275: 12737-12742Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar), very little NFκB activation was observed in the mammary glands of rats during early lactation, when type II receptor expression was lowest, but was strongly induced 2 days after removal of pups, a period of ductal involution, when type II receptor expression rebounds, indicating a correlation of NFκB activation with peripartum MIS type II receptor levels (Fig. 2 D). Because MIS-induced NFκB activation in human breast cancer cell lines led to specific up-regulation of its target gene IEX-1S, we tested whether activation of NFκB during different phases of mammary morphogenesis in the rat correlated with the levels of IEX-1 mRNA. As demonstrated in Fig. 2 E, top panels, PRG1/IEX-1S mRNA expression diminished in the lactating breast but returned to levels observed in the breasts of virgin animals after 2 days of postlactational involution, paralleling NFκB activation during this period of breast morphogenesis. Ethidium bromide staining of the gel demonstrated equal loading of RNA. PCR primers that permit the differentiation of the long and short PRG1 transcripts predominantly detected the latter in the normal breast, with a minor band of 403 base pairs (Fig. 2 E, middle panel). PRG1/IEX-1L has an in-frame insertion of 37 amino acids resulting from the presence of the entire intronic sequence within the coding region of the PRG1/IEX-1S transcript (23Wu M.X. Ao Z. Prasad K.V. Wu R. Schlossman S.F. Science. 1998; 281: 998-1001Crossref PubMed Google Scholar). To rule out the possibility that the 403-base pair band could have resulted from contaminating genomic DNA being amplified by PCR, a nonquantitative DNA amplification technique, RNase protection assay using an antisense riboprobe specific for rat PRG1/IEX-1L (Fig.2 E, Scheme), was performed. The results confirmed that PRG1/IEX-1S was the predominant transcript expressed throughout development (Fig. 2 E, bottom panel). A protected band of 403 nucleotides that would correspond to PRG1/IEX-1L was not detected. The developmental regulation of PRG1/IEX-1S coincided with NFκB DNA binding activity, suggesting that it might indeed be one of the downstream effector genes of activated NFκB in vivo in the mammary gland. Because MIS type II receptor levels, NFκB DNA binding activity, and IEX-1S expression demonstrated a compelling correlation during postnatal breast morphogenesis, we analyzed whether exogenous rhMIS could induce NFκB DNA binding activity and IEX-1S mRNA in the mammary glands of mice in vivo (n= 3). Exposure of mammary tissue to MIS resulted in the induction of NFκB DNA binding activity (Fig.3 A, top panel). Analysis of DNA-protein complexes demonstrated the presence of both p50 and p65 subunits; c-Rel was not present in the complex. The specificity of NFκB induction in vivo was demonstrated by incubating nuclear protein lysates with an oligonucleotide specific for OCT-1 (Fig. 3 A, bottom panel). These experiments identify MIS as one of the first ligands that can induce NFκB DNA binding activity in the mammary gland in vivo. The levels of circulating rhMIS in the injected animals were estimated to be 2–4 µg/ml by MIS-enzyme-linked immunosorbent assay (2Hudson P.L. Dougas I. Donahoe P.K. Cate R.L. Epstein J. Pepinsky R.B. MacLaughlin D.T. J. Clin. Endocrinol. Metab. 1990; 70: 16-22Crossref PubMed Scopus (224) Google Scholar). To determine whether MIS-mediated induction of NFκB DNA bindingin vivo correlated with gly96/IEX-1 induction, Northern blot analysis was performed. Exogenous rhMIS induced gly96/IEX-1 expression in the mammary glands of mice within 1 h of treatment compared with untreated controls and remained elevated up to 6 h (Fig.3 B, top panel). The serum rhMIS levels averaged 2–4 µg/ml in the animals. Glyceraldehyde-3-phosphate dehydrogenase levels demonstrated equal loading and indicated that the increase in gly96/IEX-1 was not due to general elevation of mRNA expression following treatment with rhMIS. Consistent with results in hum" @default.
• W2079782835 created "2016-06-24" @default.
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• W2079782835 date "2001-07-01" @default.
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• W2079782835 title "Müllerian Inhibiting Substance Regulates NFκB Signaling and Growth of Mammary Epithelial Cells in Vivo" @default.
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